Infections with the four serotypes of mosquito-borne dengue virus (DENV-1-4) are one the rise; it is currently estimated that almost 400 million people are infected with DENV annually in approximately 100 countries. Dengue disease ranges in manifestation from subclinical infection to classical dengue fever to severe disease, including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The range and incidence of severe dengue disease have risen dramatically in the last five decades. Risk of DHF/DSS is significantly elevated during a heterotypic (second serotype) secondary infection, and in part this increase in DHF/DSS is attributable in increasing co-circulation of multiple serotypes in a given locality. However each DENV serotype encompasses multiple genotypes, which differ by > 6% nucleotide identity, and genotypes in turn comprise multiple, genetically-distinct lineages, which have been termed strains, clades or groups. An increasingly common feature of DENV epidemiology is the invasion of a region by a genotype or strain of a given serotype and displacement of the native genotype or strain of that same serotype. Because genotypes and strains may differ significantly in their tendency to cause DHF/DSS during a heterotypic secondary infection or during primary infection, lineage replacement events can have a substantial impact on disease dynamics in the affected region. It is clear that a high proportion of lineage displacements in DENV are mediated by differences between lineages in intrinsic infectivity for mosquitoes, but the mechanisms by which some lineages achieve greater mosquito infectivity have not been identified. We have previously studied the infectivity of three strains of DENV-3 associated with consecutive lineage displacements in Sri Lanka. In 1989, the post-DHF strain of DENV-3 displaced the pre-DHF strain, resulting in a surge of DHF cases. In 2000, the ultra-DHF strain displaced the post-DHF strain, and DHF incidence rose again. We have shown that the post-DHF strain is more infectious than the pre-DHF strain for Ae. aegypti, but, surprisingly, that the ultra-DHF strain is less infectious for Ae. aegypti than the post-DHF strain. Building on preliminary studies in cultured mosquito cells that either possess or lack a functional RNA interference (RNAi) response, the proposed research will test the hypothesis that differences in virus strain interactions with RNAi account for the greater infectivity of the post- DHF strain relative to the pre-DHF strain but not for the greater infectivity of the post-DHF strain relative to the ultra-DHF strain. We will also seek, via transcriptome analysis, novel hypotheses for the mechanisms underpinning differences between the post-DHF and ultra-DHF strains. In the absence of a dengue vaccine or antiviral drugs, it is critical to understand the evolutionary drivers of DENV lineage displacement in order, eventually, to be able to identify and perhaps contain the spread of particularly dangerous DENV strains.